Oxidation-reduction states of FMN and FAD in NADPH-cytochrome P-450 reductase during reduction by NADPH.
NADPH-cytochrome P-450 reductase, a component of the multisubstrate monooxygenase system of liver microsomes, is an unusual flavoprotein in that it contains both FMN and FAD. In recent studies in this laboratory, a procedure was devised for selective removal of FMN from the purified enzyme, thus leading to the identification of FMN and FAD as the prosthetic groups of high and low reduction potential, respectively, and to the assignment of known reduction potentials to the individual flavin half-reactions. In the present study, the reaction of NADPH with the reductase was examined under anaerobic conditions by stopped flow spectrophotometry. The results were shown to correspond to those predicted on the basis of a model for the rapid exchange of reducing equivalents between the two flavins, the distribution being governed at any time by the reduction potentials for the individual flavin half-reactions. The reaction is divided into three steps, as follows (a) In a rapid first phase with a first order rate constant of 28 s-1, a mixture of about 70% (FMNH2, FAD) and 30% disemiquinone (FMNH ., FADH .) is generated; (FMN, FADH2), the presumed transient intermediate in the reduction of the oxidized flavoprotein by NADPH, does not accumulate under these conditions. (b) In a second phase characterized by a first order rate constant of 5.4 s-1, a mixture of 65% (FMNH2, FADH2), 24% (FMNH2, FAD), and 11% (FMNH ., FADH .) is produced. (c) Regardless of the NADPH concentration employed, a third phase occurs with very slow changes leading to an equilibrium mixture of the nine oxidation-reduction states of the reductase. The absorption spectra for all possible oxidation-reduction states of the FMN moiety of the reductase as well as of the native reductase are presented.
D003580:Cytochromes / Q000378:metabolism; D015786:Cytochromes b5; D005486:Flavin Mononucleotide / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009249:NADP / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D003580:Cytochromes; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D015786:Cytochromes b5; D009251:NADPH-Ferrihemoprotein Reductase